Production and characterization of ARO-iCre transgenic mouse line.

<p><b>(a)</b> Schematic diagram of the DNA construct used for producing ARO-iCre mice. The mammalian codon-improved Cre recombinase (iCre, 1051 bp; white box) followed by an SV40 poly(A) signal cassette (right grey box) is controlled by 304 bp of the human Cyp19/aromatase(IIa)_-278 promoter (left grey box). Arrows indicate position and direction of primers. Oligonucleotide sequences are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119392#pone.0119392.t001" target="_blank">Table 1</a>. <b>(b)</b> Cre excision is restricted to ovaries (Ov) and testes (Te) of iCre^+/0;IGF1R^flox/WT mice. The excision was only observed in gonads, not in other tissues (He, heart; Mu, muscle; Lu, lung; Sp, spleen; Br, brain; Li, liver; Pa, pancreas) from males (M) and females (F). RT-, negative control (testis cDNA without RT). PCR detected wild-type (W, 256 bp) and floxed (F, 312 bp) alleles in all tissues, and the excised allele (E, 204 bp) exclusively in gonads. <b>(c)</b> Cre recombinase activity detected in somatic cells of testes in adult ARO-iCre males. After crossing the ARO-iCre mice with a ROSA26 Cre reporter mouse, β-galactosidase activity was detected in both SC and LC. No activity was present in germ cells. Right micrograph, low magnification; left micrographs, details in higher magnification. ST, seminiferous tubules. In, interstitium. Arrow head points to SC. Bar represents 50 μm.</p

Whole testicular sperm reserve and testis weight in adult TRα^AMI-ARO, and percentage of proliferating SC in TRα^AMI-ARO at P3 <i>in vivo</i> and in testicular explants using organotypic <i>in vitro</i> cultures with or without exogenous T3.

<p>We observed a significant increase in whole testicular sperm reserve <b>(a)</b> and in testis weight <b>(b)</b> (***<i>P</i> < 0.001; n = 15 for controls and n = 16 for TRα^AMI-ARO group). Data are shown as mean ± SEM. Statistical analyses were performed using Student’s <i>t</i>-test. White bars, control; black bars, TRα^AMI-ARO (a, b). After BrdU immunohistochemical labeling, BrdU negative and BrdU positive SC were counted and the SC proliferation index was calculated. <b>(c)</b> When BrdU was injected <i>in vivo</i> 3 h before sacrifice, proliferation of Sertoli cells increased in P3 testes of TRα^AMI-ARO mice in comparison with the control (***<i>P</i> < 0.001; n = 5 animals for each genotype). <b>(d)</b> When BrdU was added <i>in vitro</i> 3 h before the end of the organotypic cultures of P3 testicular explants, the SC proliferation index significantly decreased in the control in presence of T3 compared with vehicle (***<i>P</i> < 0.001, n = 6). In contrast, T3 had no effect on SC proliferative index in TRα^AMI-ARO mice (<i>P</i> > 0.05, n = 5). Data are shown as mean ± SEM. Statistical analyses: two-way ANOVA followed by Bonferroni’s <i>post-hoc</i> test. White bars, control; black bars, TRα^AMI-ARO.</p

Plasma testosterone levels in adult TRα^AMI-ARO males, in basal conditions and after hCG stimulation, compared with control mice and mRNA levels of Cyp11a1/P450ssc, Cyp17a1/P450c17 and StAR in TRα^AMI-ARO testes.

<p><b>(a)</b> In both TRα^AMI-ARO (11 males) and controls (9 males), hCG induced a significant increase (***<i>P</i> < 0.001) in testosterone secretion, as expected. Plasma testosterone levels were unchanged in TRα^AMI-ARO compared with the control. Data are shown as mean ± SEM. Two-way ANOVA followed by Bonferroni’s <i>post-hoc</i> test. Black bars, basal levels; white bars, hCG stimulated. <b>(b)</b> The amount of cDNA of 3 steroidogenic genes (Cyp11a1/P450ssc, Cyp17a1/P450c17 and StAR) was compared in TRα^AMI-ARO (black bars) and control males (white bars). For each genotype, 6 mice were tested in triplicate using TaqMan quantitative real-time PCR, as described in materials and methods. Normalization was performed with two housekeeping genes, β-actin and GAPDH, that both exhibited similar expression among samples. mRNA expression is presented as percent of mean control level. A non-parametric test (Mann-Whitney) was used for statistical analysis. NS: not significant.</p

Primers for PCR genotyping of TRα^AMI-ARO line and for RT-PCR of TRα1 [27] and actin genes.

<p>Primers for PCR genotyping of TRα^AMI-ARO line and for RT-PCR of TRα1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119392#pone.0119392.ref027" target="_blank">27</a>] and actin genes.</p

Physiological, endocrine and cellular characteristics in TRα^AMI-ARO (present study), TRα^AMI-SC and TRα^Null/Null transgenic lines.

<p>**<i>P</i> < 0.01;</p><p>***<i>P</i> < 0.001;</p><p>ND, not determined.</p><p>Physiological, endocrine and cellular characteristics in TRα^AMI-ARO (present study), TRα^AMI-SC and TRα^Null/Null transgenic lines.</p

Histological sections of adult TRα^AMI-ARO testes at low (a) and high (b) magnification.

<p>Seminiferous tubules were fully developed in TRα^AMI-ARO. Their epithelium exhibited normal structure and organization. Hematoxylin staining. Bar represents 100 μm.</p

GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals-1

Icken while they are found on chromosome X in other depicted species. Please note that synteny is mostly conserved for bHLHPAS, 2610030H06 RIK, Mel1c, HMG2A, CD99, and myotubularin related protein in opossum and mammalian species despite the integration of new genes coding for hypothetical proteins (opossum, chimpanzee, cow), ribosomal proteins (dog, chimpanzee, man), NGFI-A binding protein (chimpanzee, man), Utbf (mouse) and MAGE (cattle) proteins. It is also of note that several genes surrounding Mel1c in zebra fish (pdcd8, nono, and the two hypothetical proteins) present high identities with genes found on chromosome X in mouse but not in the GPR50 locus (unpublished data). p.d.: predicted gene. Chrm: chromosome.<p><b>Copyright information:</b></p><p>Taken from "GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals"</p><p>http://www.biomedcentral.com/1471-2148/8/105</p><p>BMC Evolutionary Biology 2008;8():105-105.</p><p>Published online 9 Apr 2008</p><p>PMCID:PMC2323367.</p><p></p

GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals-5

APII CTD are boxed.<p><b>Copyright information:</b></p><p>Taken from "GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals"</p><p>http://www.biomedcentral.com/1471-2148/8/105</p><p>BMC Evolutionary Biology 2008;8():105-105.</p><p>Published online 9 Apr 2008</p><p>PMCID:PMC2323367.</p><p></p

GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals-3

Milarities are boxed. The positions of the transmembrane helices are reported above its sequence. Arrows indicate the positions of the amino acids that, in GPR50, evolved under positive selection.<p><b>Copyright information:</b></p><p>Taken from "GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals"</p><p>http://www.biomedcentral.com/1471-2148/8/105</p><p>BMC Evolutionary Biology 2008;8():105-105.</p><p>Published online 9 Apr 2008</p><p>PMCID:PMC2323367.</p><p></p

GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals-6

C genes and the animal orders where each branch is expressed. The trees (npl) are the fusion of three phylogenetic trees built based on Neighbour joining, maximum Parsimony and maximum Likelihood (see "Materials and Methods" section for further details). The italic letters correspond to the name given to the branches for the likelihood ratio tests (B) Phylogenetic tree of GPR50 genes. Please note that only mammalian species appear in the tree. (C) Phylogenetic tree of MT1 genes (D) Phylogenetic tree of MT2 genes. (E) Phylogenetic tree of Mel1c genes that do seem to appear only in non mammalian species. Bootstrap values are reported for each npl method.<p><b>Copyright information:</b></p><p>Taken from "GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals"</p><p>http://www.biomedcentral.com/1471-2148/8/105</p><p>BMC Evolutionary Biology 2008;8():105-105.</p><p>Published online 9 Apr 2008</p><p>PMCID:PMC2323367.</p><p></p